The involvement of dioxetane intermediates in chemiluminescent reactions was first postulated in (1968) 155. It was suggested that luciferin under the action of luciferase formed an unstable dioxetane intermediate which decomposed to produce light.
Kopecky and Mumford, Can J. Chem. (1969) 47:709 produced 3,3,4-trimethyl dioxetane which was shown to thermally decompose to acetone and aldehyde with the generation of light. Such alkyl dioxetanes are, however, too unstable at room temperature to render them useful generators of light in chemical assays.
More stable 1,2-dioxetanes were produced by replacing the alkyl groups with polycyclic hydrocarbon groups. Weiringa et al synthesized adamantlyideneadamantane 1,2-dioxetane via photooxygenation of adamantylideneadamantane, Tetrahedron. Lett. (1972) 169. This dioxetane proved to be extremely thermostable--in fact so stable that it would require detection temperatures in excess of 150.degree. C. Such elevated temperatures make the use of this dioxetane in assays of biological samples very difficult.
EPA Publication No. 0 254 051 reports the first chemically triggered 1,2-dioxetanes. See also WO 88/00695. These dioxetanes retain an adamantyl substituent on one carbon atom of the peroxide ring for stability and have an aroxy substituent protected with a group such as a phosphate, silyl, or acetyl group on the other carbon atom of the ring. These dioxetanes are sufficiently stable as long as the protecting group is present. However, once the group is removed, such as by the action of alkaline phosphatase in the case of phosphate protection or fluoride ion in the case of silyl protection, the resulting oxide intermediate is unstable and rapidly decomposes to ketones with efficient light production. While several such triggerable dioxetanes have been reported, those that provide a dioxetane that is a substrate for alkaline phosphatase (AP) have proven to be most useful both in immunoassays and in nucleic acid hybridization assays. Even though use of these AP-triggered dioxetanes has provided more sensitive assays than other nonisotopic assay formats, they still suffer from two problems. One relates to non-specific binding. The limit of detection of these assays is mostly dictated by non-specific binding of assay components leading to AP bound to surfaces independent of the presence of target molecules (noise). AP is a particularly problematic enzyme label with respect to non-specific binding. AP is ubiquitous. Its use as a specific label can be complicated by reagent and sample contamination with low levels of AP from adventitious sources. The other problem relates to the time frame of light generation. Once triggered with AP a slow (15-120 min) rate acceleration is observed followed by a constant steady state turnover (1-6 hr, depending upon the target and thus the enzyme concentration). Accordingly, at low target concentration, accumulation with an integrating detector of the total light output can take many hours. Also, there is the constant reagent-associated background light emission during the entire steady state turnover that limits detection at low non-specific binding. The present invention addresses both of these problems.